Bioadhesive Fibrin Glue
Increased experience has been gained in the use of fibrin glue among various surgical disciplines (Lerner, R. and Binur, N. S., 1990, J. Surg. Res., 48: 165-181; Gibble, J. W. and Ness, P. M., 1990, Transfusion, 30:741-747; Sierra, D. H., 1993, J. Biometer. Applic. 7, 309-352; Brennan, M., 1991, Blood Reviews, 5:240-244; Dresdale A., et al., 1985, Surgery, 97:750-755; Sponitz W., et al., 1987, Amer. Surg., 59:460-462; Schlag G. and Red1 H (Eds), 1986, Gynecology and Obstetrics-Urology. Fibrin Sealant in Operative Medicine, Vol 3, Springer Verlag (Berlin); Burnouf-Radosevich, M. et al., 1990, Vox Sang, 58:77-84). For example, surgeons, dentists and hematologists have reported that fibrin glue is an effective bioadhesive. Experience in animals and humans suggests that an advantage of using fibrin glue rather than synthetic plastics (e.g., cyanoacrylate) or sutures is that fibrin glue promotes local coagulation, thereby preventing bleeding even in hemophiliacs. Fibrin glue also appears to support regrowth of new tissue and the extracellular matrix.
Fibrin glue is formed by mixing two components, human fibrinogen (or a source of fibrinogen, such as a freeze-dried plasma protein concentrate of fibrinogen/factor XIII/fibronectin) and an activating enzyme such as thrombin. Prior to use, the plasma protein concentrates are conventionally solubilized in the presence of calcium chloride. Thrombin-induced activation of fibrinogen results in the formation of fibrin. Factor XIII and calcium participate in the cross-linking and stabilization of fibrin to become a tight mesh of polymeric fibrin glue. Applied to tissue, the fibrin clot adheres to the site of application. The rate of coagulation and mechanical properties of the clot are dependent on the concentration of fibrinogen as well as thrombin. Traditional fibrin glue preparations are described in International Application No. WO93/05067 to Baxter International, Inc.; WO92/13495 to Fibratek, Inc.; and WO91/09641 to Cryolife, Inc.
Thrombin is a common physiological instigator of clotting. Thrombin from a number of mammalian sources, most commonly bovine, is routinely used in commercially-available fibrin glues. Human thrombin can be employed in the formulation of the liposome-containing fibrin glue bioadhesive, as can other appropriate catalyzing enzymes, such as reptilase or select venoms (Fenton II, J. W. et al., 1977, J. Biol. Chem., 252:3587-3598; Gaffney P. J. et al., 1992, Thrombos. Haemostas., 67:424-427; European Patent Application No. EP 0 439 156 A1, 1991; Stocker K., et al., 1982, Toxicon., 20:265-273; Pirkle H. and Stocker K., 1991, Thrombos. Haemostas., 65:444-450).
Fibrinogen may be in an intimate admixture with other proteins that are typically found uncoagulated whole blood, in platelet-rich plasma, in plasma, in cryoprecipitate, or in precipitates of plasma obtained by a method such as Cohn precipitation of plasma. Such additional protein components may include fibronectin, immunoglobulin, particularly IgG, factor XIII, plasminogen, and albumin.
The fibrinogen preparations used in the fibrin glue and liposome compositions can be virally inactivated by one or more methods prior to their employment in the invention (e.g. Examples 1-3).
Both fibrinogen and thrombin are derived from blood plasma by the fractionation of plasma. Comprehensive reviews on the preparative techniques of each have been published and are the basis for most commercial plasma fractionation procedures used by those skilled in the art and suitable for use in the invention (For fibrinogen: Blomback, B. and Blomback, M., 1956, Ark Kemi, 10:415-443; Stryker, M. H. & Waldman, A. A., 1978, Kirk-Othmer Encyclopedia of Chemical Technology, Vol 4, 3rd ed., pp 25-61, John Wiley; Lowe G. D. O. et al., 1987, Fibrinogen 2: Biochemistry, Physiology and Clinical Relevance. Excerpta Medicus, Elsevier Science Publishers; For thrombin: Fenton II, J. W. et at., 1977, J. Biol. Chem., 252:3587-3598; Gaffney P. J. et al., 1992, Thrombos. Haemostas., 67:424-427; Ward, G., 1991, European Patent Application No. EP 0 439 156 A1; and U.S. Pat. No. 5,143,838 to Kraus et al.).
Alternative sources of human fibrinogen are also envisioned. For example, fibrinogen made by recombinant techniques could also be employed in the fibrin glue and liposome composition. Molecular techniques available for the production of recombinant fibrinogen include the use of COS-1 or Hep G2 cells transfected with DNA vectors containing isolated genes encoding normal or mutant human fibrinogen (Roy S. N. et al., 1991, J. Biol. Chem., 266:4758-4763; Roy S. N. et al., 1994, J. Biol. Chem., 269:691-695). It is expected that future developments will lead to the ability to produce usable amounts of fibrinogen by such techniques in other types of cells. Normal or mutant recombinant fibrinogens may be employed in fibrin glue compositions formulated with the types of liposomes as described herein.
Despite the effectiveness and successful use of fibrin glue by medical practitioners in Europe, neither fibrin glue nor its essential component fibrinogen is widely used in the United States at the present time because of the general risks and problems of infection from pooled blood products contaminated with lipid-enveloped viruses such as HIV, associated with AIDS, and the hepatitis-causing viruses such as HBV and HCV (also known as non A-non B hepatitis virus), as well as cytomegalovirus (CMV), Epstein-Barr virus, and the herpes simplex viruses in fibrinogen preparations. For similar reasons, human thrombin is not currently authorized for human use in the United States. Bovine thrombin, which is licensed for human use in the United States, is obtained from bovine sources which do not appear to carry significant risks for HIV and hepatitis, although other bovine pathogens may be present.
Both human fibrinogen and human thrombin can be virally inactivated against lipid coat viruses by treatment with organic solvent and detergent (SD process) (U.S. Pat. No. 4,540,573 to Neurath A. R. and Horowitz B., 1985; Horowitz, B. et al., 1985, Transfusion, 25:516-522; Horowitz, B. et al., 1992, Blood, 79:826-831; Piet, M. P. J. et al., 1990, Transfusion, 30:591-598; Burnouf-Radosevich et al., 1990, Vox Sang, 58:77-84; Horowitz, B. et al., 1992, Blood, 79:826-831). Other viral inactivation procedures for fibrinogen and thrombin blood products include UV irradiation or heating (U.S. Pat. No. 5,116,590 to Miyano, K. et al.).